Dramatic modifications and extensions of the functional properties of a transmembrane pore will be brought about by targeted modification of staphylococcal alpha-hemolysin (alphaHL). AlphaHL is secreted by Staphylococcus aureus is a water-soluble, 293-residue polypeptide, which forms heptameric pores in lipid bilayers. It is an excellent target for the proposed studies: three dimensional structures are available, the protein is robust and can be obtained in abundance, it self-assembles into membranes with a fixed subunit stoichiometry and in a single orientation, a large collection of mutants is available, the protein tolerates extreme manipulations in structure, heteroheptamers can be prepared and purified. Further, the functional properties of the pore can be examined in intricate detail by single channel recording. Given its high conductance, prolonged open stage and weak ion selectivity, alphaHL constitutes a blank slate for protein engineering. AlphaHL will be radically remodeled by targeted non-covalent and covalent modifications with cyclodextrins, cyclic peptides, responsive polymers, oligonucleotides and a bipartite chelator. The manipulations proposed, including the alteration of the inside of a protein cavity, have not been carried out previously on membrane proteins and have been performed only rarely on any class of protein. Importantly, we will not be content to demonstrate folding and assembly alone; our focus will be on function, especially alterations in unitary conductance, ion selectivity, and susceptibility to channel blockers. Expected outcomes are: (i) a better understanding of the fundamental properties of transmembrane channels; (ii) the creation of pores with new properties not found in nature; (iii) a better understanding of the rules governing the construction of modular nanostructures from biological macromolecules that do not normally interact. The work will impact healthcare, by providing new technology for biosensors, cell preservation, biotherapeutics and drug delivery.